effect of arbuscular mycorrhizal fungi on soil chemical ... ajaz malik, et al.pdf · 06.07.2018...

Int.J.Curr.Microbiol.App.Sci (2018) 7(6): 314-327

314

Original Research Article https://doi.org/10.20546/ijcmas.2018.706.036

Effect of Arbuscular Mycorrhizal Fungi on Soil Chemical Characteristics

in Apple Orchards of Kashmir Himalaya

Misbah Ajaz Malik1*

, M.Y. Zargar1, Masrat

Maqbool

2, Bisma Rashid

1,

Z.A. Baba1 and Malik Asif

1

1Division of Basic Sciences and Humanities,

2Division of Soil Sciences, Sher e Kashmir

University of Agricultural Sciences and Technology of Kashmir-193201, India

*Corresponding author

A B S T R A C T

Introduction

Arbuscular mycorrhizal fungi (AMF) are

geographically ubiquitous and occur over a

broad ecological range. They are commonly

found in association with numerous plant

species including Agricultural and

Horticultural crops. Arbuscular mycorrhizal

fungi (AMF) of the phylum Glomeromycota

are considered to associate with at least 80 per

cent of vascular land plants. The association of

plants with AMF improves plant

establishment, growth and productivity. These

fungi are well known to enhance plant nutrient

uptake, plant tolerance to drought and abiotic

stresses and to protect them against pathogens

and nematodes. Additionally, AM fungi

stabilize soils and improve soil structure

through binding sand grains and aggregate

formation.

The root infections by AMF consist of

intercellular hyphae and vesicles together with

finely branched arbuscules which develop

within the host cortical cells. External

mycelium attaches to the roots, ramifies into

the surrounding cells and produces small

vegetative spores and larger resting spores

singly or in sporocarps. The plants are

responsive to inoculation with mycorrhizal

fungi, especially in soils which are low in

fertility (Mosse, 1973; Hayman, 1982).

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 06 (2018) Journal homepage: http://www.ijcmas.com

An attempt was made to analyze the correlation between physico-chemical

factors of soil with distribution of AM fungi under temperate conditions in

different apple orchards of Kashmir Himalaya. Ten villages from district

Pulwama were selected for the present investigation because edaphic

factors of the particular district show variable soil. The study revealed that

all chemical factors viz. Organic carbon, Available nitrogen, Available

Potassium and Available phosphorus are positively correlated with

distribution of AM fungi.

K e y w o r d s

Chemical parameters,

Correlation, AM fungi,

Kashmir

Accepted:

02 May 2018

Available Online: 10 June 2018

Article Info

https://doi.org/10.20546/ijcmas.2018.706.036


315

Material and Methods

Study area

District Pulwama is an important part of

Kashmir valley with respect to the agricultural

perspective and is surrounded by Srinagar in

the north, Budgam and Poonch in the west and

Anantnag and Shopian in the east and south

side. The district is situated between 33o46´ to

33o58´ N Latitude and 74

o45´ to 75

o13´ E

longitude with a mean elevation of about 1630

m amsl.

It contributes a total geographical area of

0.109 m ha out of which 0.02365 m ha is

under agriculture and 0.0412 m ha under

forest cover, rest being used for other

purposes. Pulwama soils are shallow to deep,

mostly loam to silty loam and silty clay to

clay. The wide variations in soil

characteristics are mostly associated with

slope aspect. The soils are mostly subjected to

moderate to severe erosion and have moderate

surface stoniness at some places.

Isolation and purification of Arbuscular

mycorrhizal spores

Isolation of AM fungal spores from the

rhizospheric soil samples was done by

following Wet sieving and decanting method

(Gerdemann and Nicolson 1963). The spores

were counted under microscope Olympus

CH20i with magnification of 10×40.). Spore

population was then expressed in terms of

number of spores per 100 gm of dry soil

(Table 1). Clean and intact spores were

isolated using a specially designed needle,

spores were mounted with PVLG (poly vinyl

alcohol+ lactic acid+ glycerol) + Melzer’s

Reagent and observed under microscope and

photographed. Identification of spores up to

generic level was based on spore size, spore

colour, wall layers and hyphal attachments

using the species descriptions provided by

INVAM (http://invam.caf.wvu.edu) and other

suitable references (Schenck and Perez, 1990;

Morton and Benny, 1990; Almeida and

Schenck, 1990; Bentivenga and Morton, 1995;

Walker and Vestberg, 1998).

Assessment of AM fungal colonization of

isolated spores

The isolated spores were further purified and

mass multiplied on maize. Surface sterilized

healthy maize seeds, pre-germinated in Petri

plates under aseptic conditions, were sown in

polythene bags containing sterilized soil +

sand mixture (1:2 w/w). These bags were

aseptically inoculated with identical AM

spores at 5 cm depth (Jackson, 1973).

The bags were kept in a greenhouse at 25±3°C

and irrigated with sterile water. The plants

were uprooted after 45 days. The roots were

collected, washed with sterile water to remove

adhering soil debris and observed for

mycorrhizal infection. The infectivity was

proved by noticing the presence of hartig net,

vesicles, asbuscules or hyphae of endophytes

on roots.

For estimating mycorrhizal root colonization,

the root samples were collected and washed

carefully to remove the adhering debris. The

tertiary roots were cut into small pieces of

approximately 1cm length and subjected to

differential staining as described by (Phillips

and Hayman, 1970). The estimation of

mycorrhizal infection in roots was made by

visual observation (Giovannetti and Mosse,

1980). A randomly selected aliquot of stained

root segments, suspended in water, was spread

in a Petridish viewed under a dissecting

microscope at a magnification of 10 and 40×.

In case of AM colonization, root segments

containing vesicles and arbuscules of

endophyte and number of mycorrhizal short

roots were considered infected as suggested by

Beckjord et al., (1984).


316

Per cent mycorrhizal infection = Number of

infected root segments / Total number of

segments examined × 100

The data recorded during the investigation was

statistically analyzed with the help of Pearson

correlation (Gomez and Gomez, 1984).

Chemical parameters

The organic carbon, available nitrogen,

Available phosphorus and available K of the

soil were estimated by method of Walkey and

Black (1934), Alkaline permagnate method

(subhaiah and Asija, 1956), olsen’s method

Olsen et al., (1954) and Flamephotometric

method (Jackson, 1958) respectively.

Results and Discussion

Morphological characterization of

Arbuscular mycorrhizal spores

Spore morphology and wall characteristics

were considered for the identification of

Arbuscular mycorrhizal fungi. Four types of

genera viz., Glomus, Acaulospora,

Scutellospora and Gigaspora, were recovered

and identified. 3 to 6 unidentified spores per

gram from all studied locations were tagged as

unidentified spores (Table 2). The spore

colour of the species of Glomus was of wide

range.

It varied from red-brown to almost black or

straw to dark orange but most was yellow

brown in colour. Spores possessed globose to

sub-globose shape, about 40 to 120 µm in

size. Spore wall consisted of three layers (L1,

L2 and L3). Our findings corroborate with

those of many other workers (Koske, 1984;

Koske and Gemma, 1990). Acaulospora

spores were present singly in the soil and

develop laterally on the neck of asporiferous

saccule. Spores were light orange to yellowish

brown (Table 3 and Figures 1, 2, 3 and 4)

globose to sub-globose in shape and 150 to

210 µm in diameter. These spores were triple

layered with L1 which forms the spore surface

light yellow to apricot yellow in colour and

0.7 to 2.0 µm in thickness. L2 was laminate

and light orange to yellowish brown, 6.8 to 7.4

µm in thickness. L3 was laminate, hyaline, 0.8

to 1.6 µm in thickness and usually tightly

adherent to L2.

Similar observations have been reported by

others also (Walker et al., 2007), Sharma et

al., (2009). Scutellospora spores were with or

without ornamentations. Spores consisted of a

bilayered spore wall and two bilayered

flexible inner walls.

Thin-walled auxillary cells with smooth to

knobby surfaces were produced on hyphae in

the soil near the root surface and were also

reported by (Schenck and Perez, 1990) and

(Morton, 2002). Gigaspora spore wall

consisted of a permanent outer layer enclosing

a laminate layer, each with different properties

that distinguish species (e.g. color, thickness,

etc). Our observations corroborate with those

of Koske (1987) and Bentivenga and Morton

(1995).

There was no evidence of any ectomycorrhizal

association with apple roots, and this

corroborates with the findings of Greene et al.,

(1982). Glomus species was common and

made up for more than 75% of total isolates

followed by Acaulospora, Gigaspora and

Scutellospora.

Dominancy of Glomus in the present study is

in agreement with the findings of many other

workers (Mridha and Dhar, 2007; Burni et al.,

2009; Sharma et al., 2009). The predominance

of Glomus spp. under varying soil conditions

might be due to the fact that they are widely

adaptable to the varied soil conditions and

survive in acidic as well as in alkaline soils

(Pande and Tarafdar, 2004).


317

Root colonization studies of Arbuscular

mycorrhizal fungi

In the current study, the AM colonization in

the apple roots from Pulwama district varied

between 65.87 and 79.78% (Table 4, Figure 5,

6, 7, and 8). The results are in conformity with

the Kandula et al., (2006) who also observed

higher colonization in the apple roots and

confirmed the ubiquitous nature of AMF

spores. The highest root colonization was

recorded in response to the inoculation with

Glomus spp. (79.78%) followed by

Acaulospora species (79.56%), Gigaspora

species (73.56%) and Scutellospora species

(71.23%). Similar results were reported by

some workers Gosal et al., (2003) (Smith and

Read, 2008).

Results of the present study indicate that the

nutrient contents of the soils played a

significant role in occurrence of different

species of Arbuscular mycorrhizal fungi and it

is evident from the Perusal of the data

presented in Table 2 which revealed that AM

spore population of district Pulwama was

positively and significantly correlated with

organic carbon (r=0.887**). The results are in

conformity with those of Lipinski et al.,

(2003) who also reported a significant positive

correlation between soil organic carbon and

AM spore population.

There was a significant correlation between

AM spore population and root colonization

(r=0.512*) in district Pulwama. Kumar et al.,

(2013) also found a significant positive

correlation between mycorrhizal spores and

colonization. Yang et al., (2010) found a

positive correlation between and mycorrhizal

colonization and spores. These results are also

supported by Li et al., (2009). The positive

and significant correlation between AM spores

and available nitrogen (r=0.815*) was found

which might be due to the fact that nitrogen

and organic carbon are required by micro-

organisms for their special requirements and

as a result high nitrogen and organic carbon in

the soil increased infection and population of

AM fungi. Similar results were reported by

Venkatrao et al., (1972).

Since the climatic conditions of the study area

fall under temperate zone which are conducive

to the mycorrhizal development, it is possible

that concentration of such propagules may be

higher (Akhter, 2005)

Moreover, influence of apple roots through

their exudates cannot be ruled out which needs

further studies.

Chemical analysis of soil

Organic carbon

Perusal of the data presented in Table 5

indicates that organic carbon content of soils

of identified villages ranged between 1.68%

(Shiekhar) and 1.96% (Pinglin) with an

average of 1.80%. The organic carbon content

of Pinglin soil was significantly more than the

soils of other villages except for Rajpora and

Shiekhar which was statistically at par. Najar

(2002) found that organic carbon content

varied from 1.4 to 2.4, 0.9 to 1.8 and 0.5 to 1.2

in the high altitude and valley basin orchard

soils of Kashmir. Wani et al., (2010) reported

organic carbon content of 1.1 to 2.10% in the

soils of Himalayan region. The variation in the

organic carbon content may be due to

variation in the application of organic manures

and the population of the soil microorganisms.

As a food source for soil fauna and flora, soil

organic carbon plays an important role in the

soil food web by controlling the number and

types of soil inhabitants which serve important

functions such as nutrient cycling and

availability, assisting root growth and plant

nutrient uptake, creating burrows and even

suppressing crop diseases. These findings are

supported by Bulluck et al., (2002).


318

Table.1 Mean Spore count per gram of rhizosphere soil of District Pulwama

Villages Spore population/g

Rajpura 7

Shadimarg 12

Nikas 7

Drubgam 7

Shiekar 8

Tikkin 10

Sunsomil 8

Pinglin 12

Gungoo 8

Puhoo 8

Mean 8.7

Table.3 Morphological features of isolated genera of AM fungi

Genera Spore size

(µm diameter)

Spore shape Spore colour Spore wall Hyphal

colour

Acaulospora 115-170 Globose to

sub globose

Yellow brown to dark

brown

Three layered

(L1.L2 and L3)

Grey

white

Gigaspora 200-300 Globose to

sub globose

White to cream usually

a rose pink tint.

Bilayered layered

(L1 and L2)

Orange

brown

Scutellospora 100-170 Sub globose

to ellipsoid

to oblong

Cream to yellow or

pale orange brown to

dark orange brown

Bilayered spore

wall (L1 and L2)

Hyaline

to orange

white.

Glomus 40 – 120 Globose to

ellipsoid

red brown to almost

black most are yellow

brown

three layered

(L1,L2 and L3)

Hyaline

to

yellowish.

Table.2 Isolation of Arbuscular mycorrhizal spores from rhizospheric soil of apple

from different locations of District Pulwama

Locations Spore count per gram of soil (Identified Genera)

Acaulospora Scutellospora Gigaspora Glomus Unidentified

Genera

Rajpora - - 1 2 4

Shadimarg 2 - 3 3 4

Nikas 1 2 - - 4

Drubgam - 1 1 2 3

Shiekar 2 - 1 - 5

Tikkin 3 - - 3 4

Sunsomil - - 2 2 4

Pinglin 3 2 1 - 6

Gungoo - 1 - 4 3

Puhoo 3 - - - 5


319

Table.4 In vitro root colonization by AM fungal spores isolated from District Pulwama

Locations Genera Root colonization (%)

Rajpora Gigaspora sp.

Glomus sp.

68.23

72.05

Shadimarg Acaulospora sp.

Gigaspora sp.

Glomus sp.

65.87

70.03

72.13

Nikas Scutellospora sp.

Acaulospora sp.

69.09

70.05

Drubgam Scutellospora sp.

Glomus sp.

Gigaspora sp.

71.23

76.67

69.08

Shiekar Gigaspora sp.

Acaulospora sp.

73.56

68.67

Tikkin Acaulospora sp.

Glomus sp.

72.13

68.78

Sunsomil Gigaspora sp.

Glomus sp.

67.67

79.55

Pinglin Acaulospora sp.

Gigaspora sp.

Scutellospora sp.

79.56

67.80

65.87

Gungoo Scutellospora sp.

Glomus sp.

69.77

79.78

Puhoo Acaulospora sp. 76.86

Table.5 Chemical characteristics of soil samples of District Pulwama

Villages Or.

Carbon

(%)

Av.

Nitrogen

(kg/ha)

Av.

Phosphorus

(kg/ha)

Av.

Sulphur

(Kg/ha)

Av.

Potassium

(kg/ha)

Rajpura 1.74 352.23 16.21 11.23 186.01

Shadimarg 1.77 359.04 17.63 11.92 188.20

Nikas 1.93 370.01 16.43 12.51 186.11

Drubgam 1.79 365.34 17.34 12.01 188.23

Shiekar 1.68 343.12 17.98 11.61 183.65

Tikkin 1.83 360.05 17.23 12.13 188.06

Sunsomil 1.74 351.23 17.01 11.61 180.13

Pinglin 1.96 385.12 17.63 12.90 189.12

Gungoo 1.82 358.07 17.60 12.02 188.14

Puhoo 1.75 356.12 17.62 11.74 188.02

Mean 1.801 360.033 17.268 11.968 186.567

CD (p≤0.05) 0.069 2.589 0.306 0.702 NS

CV 2.22 8.416 1.025 3.428 1.679


320

Table.6 Correlation between spore population and other studied

parameters of District Pulwama

Parameters Spore population

Spore population 1

Organic carbon 0.887**

Available Nitrogen 0.815*

Available Phosphorus 0.797

Available Sulphur 0.910

Available Potassium 0.614*

Root colonization 0.512*

*. Correlation is significant at the 0.05 level

**. Correlation is significant at the 0.01 level

Table.7 Correlation between available nutrients of representative

samples in District Pulwama

Nutrients Organic

Carbon

Available

Nitrogen

Available

Phosphorus

Available

Sulphur

Available

Potassium

Organic carbon 1 0.931** 0.514 0.923** 0.784*

Available

Nitrogen

0.931** 1 0.575 0.803** 0.583

Available

Phosphorus

0.514 0.575 1 0.406 0.530

Available

Sulphur

0.923** 0.803** 0.406 1 0.493

Available

Potassium

0.784* 0.583 0.530 0.493 1

*. Correlation is significant at the 0.05

**. Correlation is significant at the 0.01 level

Study area


321

Fig.1 Spores of Acaulospora

Fig.2 Spore of Glomus

Fig.3 Spores of Gigaspora


322

Fig.4 Spores of Scutellospora

Fig.5 Root colonization of the genus Acaulospora

Fig.6 Root colonisation of the genus Scutellospora

Fig.7 Root colonisation of the genus Glomus


323

Fig.8 Root colonisation of the genus Gigaspora

Available nitrogen

Available nitrogen content of the soils ranged

from 343.12 kg ha-1

(Shiekhar) and 385.12 kg

ha-1

(Pinglin) with an average value of 360.03

kg ha-1

. The available nitrogen content of

Pinglin soil was significantly more than soils

of other villages. However, available nitrogen

of soils from Shadimarg and Gungoo were

statistically at par. This is supported by the

findings of Dar (1996). Najar (2002) also

observed the available nitrogen content in the

range of 200 to 350 kg/ha. Bhat (2001) while

studying the apple orchard soils of north

Kashmir also reported that, the available

nitrogen content ranged from 300 to 410

kg/ha. Akhtar (2005) while working in the

apple orchard soils of Kashmir valley also

reported the available nitrogen content in the

range of 320 to 410 kg/ha.

Available phosphorus

Available phosphorus content of soils ranged

between 16.21 kg ha-1

(Rajpura) and 17.98 kg

ha-1

(Shiekhar) with an average value of 17.26

kg ha-1

. The available phosphorus content of

Shiekhar soil was significantly more than

soils of other villages except Shadimarg and

Pinglin which were statistically at par. Khan

et al., (2013) reported that the available

phosphorus was in the range of 10 to 24

kg/ha. The high available phosphorus may be

due to application of organic manures like

FYM, vermicompost and biofertlizer. These

findings are supported by Kumar et al.,

(2009).

Available potassium

Available potassium content of soils ranged

between 180.13 kg ha-1

(Sunsomil) and

189.12 kg ha-1

(Pinglin) with a mean value of

186.56 kg ha-1

. This is supported by the

findings of Aon and Colaneri (2001) who also

observed the potassium in range of 178.67 to

197.12kg/ha in the apple orchard soils of

Himachal Pradesh.

Correlation studies

Table 6 revealed that correlation of AM spore

population with organic C (r=0.887**),

available N (r=0.815*), available K

(r=0.614*) and root colonization (r=0.512*)

were found to be positive and significant in

District. However positive, but non-

significant correlation was observed with

respect to available P. The results are in

conformity with those of Lipinski et al.,

(2003) who also reported a significant

positive correlation between soil organic

carbon and AM spore population. The

positive and significant correlation between

AM spores and available nitrogen (r=0.815*)

was found which might be due to the fact that

nitrogen and organic carbon are required by

micro-organisms for their special


324

requirements and as a result high nitrogen and

organic carbon in the soil increased infection

and population of AM fungi. Similar results

were reported by Venkatrao et al., (1972).

Between available nutrients of

representative samples of District Pulwama

Perusal of the data present in Table 7 reveals

that the organic carbon content of soils of

representative villages was found to be


available nitrogen (r=0.931**), available

sulphur (r=0.923**) and available potassium

(r=0.784*) but correlation was positive and

non-significant with available phosphorus.

The available nitrogen content of soils was


organic carbon and available S while as

positive and non-significant correlation was

observed with available P and K. The

available phosphorus content of soils was

positive and non-significantly correlated with

Organic C, available N, S and K contents. The

available sulphur from soils was found to be


organic carbon (r=0.923**), available N

(r=0.803**) while as positive and non-

significant correlation was recorded with

Available P and K. The available potassium

was found to be positively and non-

significantly correlated with available N, P

and S from the soils. However, the correlation

with organic carbon (r=0.784*) was positive

and significant.

Acknowledgement

The authors are thankful to Sher e Kashmir

University of Agricultural Sciences and

Technology of Kashmir for providing

laboratory facilities for carrying out this

research work. The help rendered by

orchardists during field surveys are highly put

on record.

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How to cite this article:

Misbah Ajaz Malik, M.Y. Zargar, Masrat Maqbool, Bisma Rashid, Z.A Baba and Malik Asif.

2018. Effect of Arbuscular Mycorrhizal Fungi on Soil Chemical Characteristics in Apple

Orchards of Kashmir Himalaya. Int.J.Curr.Microbiol.App.Sci. 7(06): 314-327.

doi: https://doi.org/10.20546/ijcmas.2018.706.036

https://doi.org/10.20546/ijcmas.2018.706.036

effect of arbuscular mycorrhizal fungi on soil chemical ... ajaz malik, et al.pdf · 06.07.2018...

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